温度对双低两用核不育水稻96-5-2S与培矮64S育性的影响

在自然变温、人工控温及冷水灌溉条件下,比较研究了温度对双低两用核不育水稻96－5－2S与两用核不育水稻培矮64S育性影响的差异。结果表明：（1）当它们在雄性育性转换温敏感期1－12d平均自然日均温23.0-23.8℃的低温时,96－5－2S表现不良,套袋自交结实率为0,而培矮46S可育,套袋自交结实率为0．1％－4．5％;（2）在它们雄性育性转换温敏感期用22℃恒温处理5d,96－5－2S败育彻底,套袋自交结实率为0,而培矮64S可育,套袋自交结实率为10．7％;用17℃恒温处理6d,96－5－2S与培矮64S均可育,但96－5－2S套袋自交结实率（6．8％）显著高于培矮64S（2．5％）;（3）在它们雄性育性转换温和不同温度的冷水串灌15d,水深维持在20cm左右,当水温为22－22．5℃时,96－5－2S不育,结实率为0,而培矮64S可育,结实率为18．5％;当水温为19．5－21．5℃时,96－5－2S与培矮64S均可育,但96－5－2S结实率（2．5％－45．1％）显著或极显著低于培矮64S（50．4％－56．9％）以上结果说明：导致双低两用核不育水稻96－5－2S雄性不育的起点温度与导致其生理不育的下限温度均低,其不育性比培矮64S更稳定,耐寒性比培矮64S更强,即可确保制种安全,又可确保自身繁殖,对加快两系法杂交水稻的发展步伐将起到重要的促进作用。     

Characterization of an RNase Z nonsense mutation identified exclusively in environment-conditioned genic male sterile rice

The two-line hybrid system in rice is becoming more important and employs environment-conditioned genic male sterile (EGMS) lines sensitive to photoperiod (photoperiod-sensitive genic male sterile), temperature [temperature genic male sterile (TGMS)], or a combination of the two (photoperiod temperature genic male sterile). At least 18 EGMS genes have been mapped, and two cloned, but controversies exist. For example, three different genes were reported to underlie the TGMS trait in three independently identified progenitors, Annong S-1, Zhu 1S, and Guangzhan 63S, while another study demonstrated that the TGMS genes in Annong S-1 and Zhu 1S are allelic. In the present study, we confirmed the allelism of the three TGMS genes, which means there is a common TGMS gene(s) in these lines. Knowing there is an association between the mutant allele (RNZ ^m ) of a ribonuclease gene (RNZ) with the TGMS trait in Guangzhuan 63S, we then sequenced RNZ for 14 commercial EGMS and 21 non-EGMS lines, and we developed two derived cleaved amplified polymorphic sequence (dCAPS) markers to detect RNZ ^m alleles in 32 EGMS and 310 non-EGMS lines. The analyses showed that the RNZ ^m allele existed exclusively in EGMS lines; all non-EGMS lines contained the functional RNZ ^gc or RNZ ^tc allele. Furthermore, two segregating populations that included 2,429 individuals were developed by crossing Zhu 1S (RNZ ^m ) to two non-EGMS lines (both with RNZ ^tc ); examination of the segregation of male sterile and fertile plants indicated that the TGMS trait was under the control of a single gene; analysis of the markers revealed the RNZ ^m allele exclusively in TGMS plants and the RNZ ^tc allele only in non-TGMS plants in both populations. The dCAPS markers could therefore help select TGMS progeny in breeding programs, which will save time and labor, and improve breeding efficiency and accuracy.     

Aanlysis of short photo-periodic sensitive genic male sterility and molecular mapping of rpms3(t) gene in rice (Oryza sativa L.) using SSR markers

A research was conducted on the pollen fertility of rice sterile lines D52S and D38S responsive to photoperiod during the sensitive stage under natural and controlled conditions. Bulk segregant analysis (BSA) and recessive class approach were applied to identify DNA markers that co-segregate with gene conferring male-sterility in D52S mutant rice. The results showed that in day-light higher or equal to 14.00 h, D52S and D38S rice pollen were fertile; however, they were sterile when day-length was less than 14.00 h. They were therefore considered to be short photo-periodic sensitive genic male sterile lines(Short PGMS lines). Under short day-light conditions, the pollen fertility segregation of F₂ populations from crosses between D52S/Shuhui527 and D52S/Gui99showed 3:1 ratio of fertile to sterile plants suggestingthat male sterility in D52S was controlled by one recessive gene. Two markers RM244 and RM216 located on chromosome number 10 co-segregated completely with the rpms locus. The locus was mapped to the interval between SSR markers RM2571 (6.6 cM) and RM244 (4.6 cM).     

Photoperiod- and thermo-sensitive genic male sterility in rice are caused by a point mutation in a novel noncoding RNA that produces a small RNA

Photoperiod- and thermo-sensitive genic male sterility (PGMS and TGMS) are the core components for hybrid breeding in crops. Hybrid rice based on the two-line system using PGMS and TGMS lines has been successfully developed and applied widely in agriculture. However, the molecular mechanism underlying the control of PGMS and TGMS remains obscure. In this study, we mapped and cloned a major locus, p/tms12-1 (photo- or thermo-sensitive genic male sterility locus on chromosome 12), which confers PGMS in the japonica rice line Nongken 58S (NK58S) and TGMS in the indica rice line Peiai 64S (PA64S, derived from NK58S). A 2.4-kb DNA fragment containing the wild-type allele P/TMS12-1 was able to restore the pollen fertility of NK58S and PA64S plants in genetic complementation. P/TMS12-1 encodes a unique noncoding RNA, which produces a 21-nucleotide small RNA that we named osa-smR5864w. A substitution of C-to-G in p/tms12-1, the only polymorphism relative to P/TMS12-1, is present in the mutant small RNA, namely osa-smR5864m. Furthermore, overexpression of a 375-bp sequence of P/TMS12-1 in transgenic NK58S and PA64S plants also produced osa-smR5864w and restored pollen fertility. The small RNA was expressed preferentially in young panicles, but its expression was not markedly affected by different day lengths or temperatures. Our results reveal that the point mutation in p/tms12-1, which probably leads to a loss-of-function for osa-smR5864m, constitutes a common cause for PGMS and TGMS in the japonica and indica lines, respectively. Our findings thus suggest that this noncoding small RNA gene is an important regulator of male development controlled by cross-talk between the genetic networks and environmental conditions.     

Male-sterility of thermosensitive genic male-sterile rice is associated with premature programmed cell death of the tapetum

The tapetum plays a crucial role in pollen development. This secretory tissue produces numerous nutritive proteins necessary for pollen maturation. The tapetum, whose cells undergo programmed cell death (PCD), is completely diminished by the time the pollen is fully mature. Our previous studies on a thermosensitive genic male-sterile (TGMS) rice (Oryza sativa L.) suggested that male-sterility was due to failure in pollen development. In this paper we describe how further analysis of the TGMS rice revealed that male-sterility is associated with premature PCD of the tapetum. Cytological observations of TGMS rice anthers at various developmental stages indicated that PCD initiates at an early stage of pollen development and continues until the tapetal cells are completely degraded, resulting in pollen collapse. Transmission electron microscopy showed the morphologically distinct hallmarks of apoptosis, including cytoplasmic shrinkage, membrane blebbing, and vacuolation. Identification of DNA fragmentation using the TUNEL assay supports the hypothesis that premature PCD is associated with male-sterility in the rice. The tissue-specific feature of the thermosensitive genic male-sterile phenotype is discussed with regard to PCD during anther development.     

Effect of Growth Regulators and Chemicals on Pollen Sterility in TGMS Lines of Rice

Thermosensitive genic male sterility (TGMS) in rice is a widely adopted technique for successful hybrid rice production in Asia. TGMS lines remain male sterile when daily mean temperature is above the critical sterility temperature and are therefore used as female parents. The same line will remain fertile when mean temperature is below the critical sterility temperature. Achievement of 100% male sterility in TGMS lines is important for the successful utilization of TGMS lines as female parents in hybrid rice production. This study examined the external application of some growth regulators and chemicals and their effect on pollen sterility. Among the various treatments, ethrel (800 ppm), salicylic acid (600 ppm) and maleic hydrazide (0.2%) induced a significantly higher percentage of male sterility in the TGMS lines. The sprayed plants also showed higher total phenol accumulation in their flag leaves. The results suggest that it is possible to achieve 100% male sterility in TGMS lines with the external application of growth regulators and chemicals.     

Molecular mapping of two reverse photoperiod-sensitive genic male sterility genes (rpms1 and rpms2) in rice (Oryza sativa L.)

The reverse photoperiod-sensitive genic male sterility (PGMS) and thermo-sensitive genic male sterility (TGMS) lines have an opposite phenotype compared with normal PGMS and TGMS lines widely used by the two-line system in current hybrid rice seed production. Thus, the application of reverse PGMS and TGMS lines can compensate PGMS and TGMS lines in hybrid rice production. YiD1S is a reverse PGMS line, in which pollen fertility is mainly regulated by day-length, but also influenced by temperature. Genetic analysis indicated that male sterility of YiD1S was controlled by two recessive major genes. An F₂ population from a cross between YiD1S and 8528 was developed and used for molecular mapping of the two reverse PGMS genes which were first named rpms1 and rpms2. Both simple sequence repeat (SSR) markers and bulked segregant analysis (BSA) were used in this study. As a result, one reverse PGMS gene (rpms1) was mapped to the interval between SSR markers RM22980 (0.9 cM) and RM23017 (1.8 cM) on chromosome 8. Eight SSR markers, YDS818, RM22984, RM22986, RM22997, YDS816, RM23002, RM339 and YDS810 completely co-segregated with the rpms1 gene. Another reverse PGMS gene (rpms2) was mapped to the interval between SSR markers RM23898 (0.9 cM) and YDS926 (0.9 cM) on chromosome 9. The physical mapping information from publicly available resources shows that the rpms1 and rpms2 loci are located in a region of 998 and 68 kb, respectively. The analysis based on marker genotypes showed that the effect of rpms1 was slightly larger than that of rpms2 and that the two genes interacted in controlling male sterility. H. F. Peng, Z. F. Zhang and B. Wu contributed equally to this work.     

光照强度对温光敏细胞核雄性不育小麦育性的影响

以具有低温不育、高温可育特性的温光敏细胞核雄性不育小麦C412S和C404S为材料,以其回交转育亲本、育性正常的C412和C404为对照,用人工气候箱研究了光照强度对温光敏细胞核雄性不育小麦育性表达的影响。结果表明,在低温条件下(日温8℃/夜温6℃),C412S和C404S在不同光照强度(160μmol/m2.s和300μmol/m.2s)下自交结实率都为0,表现为完全不育。在较高温度条件下(日温18℃/夜温14℃),从花粉母细胞形成期到开花期的光照处理,C412S在160μmol/m.2s弱光照下的自交结实率仅为5.4%,表现为高不育,在300μmol/m2.s较强光照下的自交结实率高达65.0%,表现为高度可育;而另一不育系C404S在2种光照强度下的自交结实率分别为69.9%和73.2%,都达到了高度可育水平。表明光照强度对温光敏细胞核雄性不育小麦的雄性育性表达具有重要影响,但不同材料对光照强度的响应程度有所差异。     

Comparative proteomics analysis reveals the mechanism of fertility alternation of thermosensitive genic male sterile rice lines under low temperature inducement

Thermosensitive genic male sterile (TGMS) rice line has made great economical contributions in rice production. However, the fertility of TGMS rice line during hybrid seed production is frequently influenced by low temperature, thus leading to its fertility/sterility alteration and hybrid seed production failure. To understand the mechanism of fertility alternation under low temperature inducement, the extracted proteins from young panicles of two TGMS rice lines at the fertility alternation sensitivity stage were analyzed by 2DE. Eighty‐three protein spots were found to be significantly changed in abundance, and identified by MALDI‐TOF‐TOF MS. The identified proteins were involved in 16 metabolic pathways and cellular processes. The young panicles of TGMS rice line Zhu 1S possessed the lower ROS‐scavenging, indole‐3‐acetic acid level, soluble protein, and sugar contents as well as the faster anther wall disintegration than those of TGMS rice line Zhun S. All these major differences might result in that the former is more stable in fertility than the latter. Based on the majority of the 83 identified proteins, together with microstructural, physiological, and biochemical results, a possible fertile alteration mechanism in the young panicles of TGMS rice line under low temperature inducement was proposed. Such a result will help us in breeding TGMS rice lines and production of hybrid seed.     

Strategy and utilization of a herbicide resistance gene in two-line hybrid rice

Hybrid rice plays an important role in China's aim to improve rice production as it accounts for some 50% of rice planting area but produces about 60% of the total rice grain. However, the existing three-line system used in hybrid rice production has its limitations. The two-line system, which makes use of photoperiod-sensitive genic male-sterile (PGMS) and thermo-sensitive genic male-sterile (TGMS) lines to generate the male-sterile parental line, was developed to overcome some of these limitations. The sterility of the male-sterile line of two-line hybrid rice, however, fluctuates when the temperature-sensitive phase of fertility encounters abnormal low temperatures during hybrid seed production, which induces selfing and decreases the purity of hybrid. We describe here the strategy of utilizing a herbicide resistance gene in two-line hybrid rice to eliminate this fluctuation in the sterility of the P/TGMS lines during hybrid seed production and reports the development of the herbicide resistance restorer line Bar68-1 and its herbicide-resistant early season hybrid rice Xiang125s/Bar68-1. When the restorer line and its derived hybrid are herbicide resistant, the selfed seeds can be removed easily from the hybrid by herbicide spraying. A herbicide resistance gene bar was transferred into a restorer line by particle bombardment. The resulting transgenic restorer line Bar68-1 and its hybrid Xiang125 s/Bar68-1 inherited stable herbicide resistance. The purity of Xiang125s/Bar68-1 was increased by spraying the seed bed with herbicide, which resulted in a significant increase in yield, grain quality, and disease resistance in comparison to the controls in a regional trial.     

Genetic characterization and fine mapping of a novel thermo-sensitive genic male-sterile gene tms6 in rice (Oryza sativa L.)

The application of genetic male sterility in hybrid rice production has great potential to revolutionize hybrid seed production methodology. The two-line breeding system by using thermo-sensitive genic male sterility (TGMS) has been discovered and successfully developed as a breeding strategy in rice. One TGMS gene was investigated by a spontaneous rice mutant line, Sokcho-MS, originated from a Korean japonica variety. It was shown that Sokcho-MS is completely sterile at a temperature higher than 27°C and/or lower than 25°C during the development of spikelets, but fertile at the temperature ranging from 25 to 27°C regardless of the levels of day-length. Genetic analysis and molecular mapping based on SSR, STS and EST markers revealed that a single recessive gene locus involved the control of genic male sterility in Sokcho-MS. By using an F₂ mapping population derived from a cross between Sokcho-MS and a fertile indica variety Neda, the new TGMS gene, designated as tms6, was mapped primarily to the long arm of chromosome 5 of Oryza sativa at the interval between markers E60663 (2.0 cM) and RM440 (5.8 cM). Subsequently, tms6 was fine mapped to the interval between markers RM3351 (0.1 cM) and E60663 (1.9 cM). As tms6 appeared to be independent of other mapped TGMS genes in rice, the genetic basis of Sokcho-MS was further discussed.     

Differentiation and classification of parental lines and favorable genic interactions affecting F1 fertility in distant crosses of rice (Oryza sativa L.)

 This study was intended to investigate the extent of genetic differentiation in parental lines of rice hybrids and to analyze the genetic basis underlying the fertility phenomenon in distant crosses. Two subsets of rice material (111 entries in total) were used, including 81 doubled-haploid (DH) lines and 30 Indica and Japonica rice varieties or lines (as a control). The DH lines was derived from a heterotic Indica/Japonica cross (Gui630/02428) by anther culture. The materials in the control represent a broad spectrum of the Asian cultivated rice gene pool including landraces, primitive cultivars, historically important cultivars, modern elite cultivars, super rice and parents of superior hybrids. In accordance with the NC II design, 57 out of the DH lines were test-crossed to two important wide compatibility lines: photoperiod-sensitive genetic male sterile (PGMS) line N422s and thermo-sensitive genetic male sterile (TGMS) line Peiai64s. The F₁s and their parents, 182 entries in total, were examined for the performance of seven traits in a replicated field trial. All the rice materials was surveyed for polymorphisms using 92 RFLP markers selected from two published molecular marker linkage maps. Genotypes of the F₁ hybrids at the molecular-marker loci were deduced from the parental genotypes. The analysis showed that there were two types of genetic differentiation in the two subsets of rice material; that is, qualitative differentiation in the control and quantitative differentiation in the DH lines. In addition, favorable genic interactions (both intra- or inter-locus) contributed to better increase the fertility in hybrids of distant crosses through incorporation of a wide-compatibility line as the female parent. Favorable genic interactions can be applied in hybrid rice breeding programs by selecting parents with an appropriate extent of genetic differentiation. Received: 5 June 1997 / Accepted: 10 September 1997     

The extent of parental genotypic divergence determines maximal heterosis by increasing fertility in inter-subspecific hybrids of rice (Oryza sativa L.)

The magnitude of heterosis in F₁ hybrids is related not only to the performance of parents per se but also to the genetic diversity between two parents. The extent of genotypic divergence between hybrid rice parents was investigated at the molecular level, using two subsets of rice materials: a subset of doubled haploid (DH) lines derived from an Indica × Japonica cross (Gui630/02428) and another subset of Indica or Japonica lines representative of a broad spectrum of the Asian cultivated rice gene pool, including landraces, primitive cultivars, historically important cultivars, modern elite cultivars, super rice and parents of superior hybrids. 57 entries deliberately selected from the 81-DH lines (in total) were testcrossed to two widely used rice lines in China, photoperiod-sensitive genic male sterile (PGMS) N422s and thermo-sensitive genic male sterile (TGMS) Peiai64s. Results of the two sets of test-cross F₁ populations showed congruently that parental genotypic divergence has a relatively low impact on heterosis for the two yield components, i.e., panicle number and 1000-grain weight, but it has a great bearing on fertility parameters, i.e., filled grains per plant and seedset. Heterosis for grain yield in the two test-cross populations exhibited a sharp maximum when the proportion of Japonica alleles in the male parent was between 50 and 60%, so was the heterosis for fertility parameters correspondingly. Thus fertility parameters were the most sensitive and important factors which were influenced by the extent of parental genotypic divergence. Moreover, our results showed that parents with moderate extent of genotypic divergence played an important role in the use of inter-subspecific rice heterosis.     

主要作物光温敏核雄性不育基因的研究进展与应用*

光温敏核雄性不育系在不同的生态环境条件下可以实现一系两用,简化制种程序,是农作物杂交种子生产的一种重要资源。简要介绍了主要作物杂交种子生产方式,综述了水稻、小麦、玉米、谷子等作物光温敏核雄性不育系的研究进展以及在两系杂交种子生产上的应用,并探讨了光温敏核雄性不育系的应用前景。     

Temperature during the day, but not during the night, controls flowering of Phalaenopsis orchids

Phalaenopsis orchids are among the most valuable potted flowering crops commercially produced throughout the world because of their long flower life and ease of crop scheduling to meet specific market dates. During commercial production, Phalaenopsis are usually grown at an air temperature > or =28 degrees C to inhibit flower initiation, and a cooler night than day temperature regimen (e.g. 25/20 degrees C day/night) is used to induce flowering. However, the specific effect of day and night temperature on flower initiation has not been well described, and the reported requirement for a diurnal temperature fluctuation to elicit flowering is unclear. Two Phalaenopsis clones were grown in glass greenhouse compartments with constant temperature set points of 14, 17, 20, 23, 26, or 29 degrees C and fluctuating day/night (12 h/12 h) temperatures of 20/14, 23/17, 26/14, 26/20, 29/17, or 29/23 degrees C. The photoperiod was 12 h, and the maximum irradiance was controlled to < or =150 micromol m(-2) s(-1). After 20 weeks, > or =80% of plants of both clones had a visible inflorescence when grown at constant 14, 17, 20, or 23 degrees C and at fluctuating day/night temperatures of 20/14 degrees C or 23/17 degrees C. None of the plants were reproductive within 20 weeks when grown at a constant 29 degrees C or at 29/17 degrees C or 29/23 degrees C day/night temperature regimens. The number of inflorescences per plant and the number of flower buds on the first inflorescence were greatest when the average daily temperature was 14 degrees C or 17 degrees C. These results indicate that a day/night fluctuation in temperature is not required for inflorescence initiation in these two Phalaenopsis clones. Furthermore, the inhibition of flowering when the day temperature was 29 degrees C and the night temperature was 17 degrees C or 23 degrees C suggests that a warm day temperature inhibits flower initiation in Phalaenopsis.     

Developing disease‐resistant thermosensitive male sterile rice by multiplex gene editing

High-quality and disease-resistant male sterile lines have great potential for applications in hybrid rice breeding. We introduced specific mutations into the TMS5, Pi21, and Xa13 genes in Pinzhan intermediate breeding material using the CRISPR/Cas9 multiplex genome editing system. We found that the transgenefree homozygous triple tms5/pi21/xa13 mutants obtained in the T1 generation displayed characteristics of thermosensitive genic male sterility(TGMS) with enhancedresistance to rice blast and bacterial blight. Our study provides a convenient and effective way of converting breeding intermediate material into TGMS lines through multiplex gene editing, which could significantly accelerate the breeding of sterile lines.